Because apoptosis, a biochemically distinct form of cell death, is one of the major responses triggered in cancer cells by either chemotherapy or hormonal manipulation, there has been substantial interest in understanding regulation of the apoptotic process. Studies performed over the past two decades have demonstrated that Bcl-2 family members play critical roles in cellular life/death decisions. In particular, it is thought tat Bax and Bak cause mitochondrial outer membrane permeabilization (MOMP); antiapoptotic family members such as Bcl-2 inhibit MOMP; and members of the BH3-only subfamily facilitate MOMP by either binding to Bax/Bak (direct activators) or binding the antiapoptotics (sensitizers). How Bax and Bak are activated and how they cause MOMP is incompletely understood. In preliminary studies, we have examined BH3-only protein-mediated Bak activation. Using purified proteins, we have shown for the first time that Bak oligomerization is a two-step process involving initial formation of a BH3 protein*Bak heterodimer followed by release of the BH3-only protein during formation of Bak homo-oligomers. We have also mapped the interacting domains on the BH3-only protein and Bak and have confirmed their involvement during Bak activation in a cellular context. Importantly, the results of this analysis suggest substantial differences between Bak oligomerization and each of the current models of Bax activation. Moreover, our results implicate Noxa, like Bim and tBid, as an activator BH3-only protein. To build on our results, we now propose to 1) examine the effect of BH3-only peptide binding on the Bak monomer, thereby confirming the putative binding site and providing insight into the structural changes that prepare Bak for oligomerization; 2) elucidate the structure of Bak oligomers through a combination of morphological, biochemical and computational approaches designed to provide new information about the action of Bak and the process of MOMP; and 3) examine posttranslational regulation of Noxa, which we have identified as a critical participant in Bak activation during bortezomib-induced apoptosis in lymphoid cells. If successful, the proposed experiments will not only provide new insight into the process of Bak activation, but also yield important new information about action of an increasingly widely used anti-lymphoma agent. PUBLIC HEALTH RELEVANCE: A substantial fraction of the 1.4 million Americans diagnosed with cancer in 2010 will receive chemotherapy and/or hormonal therapy. One of the major outcomes of this therapy, when successful, is the induction of apoptosis in susceptible cancer cells. A group of proteins called Bcl-2 family members play critical roles in determining whether cells undergo apoptosis. In particular, the proteins Bax and Bak are thought to trigger the death process by punching holes in an intracellular boundary called the outer mitochondrial membrane, thereby activating biochemical changes that ultimately lead to apoptosis. How Bax and Bak are activated, and how they subsequently permeabilize membranes, is incompletely understood. Building on earlier observations, we have recently developed a method for studying Bak activation and membrane permeabilization using highly purified proteins. We now propose to characterize the activated Bak molecules, study the Bak activation process, and explore the chemotherapy-modulated regulation of Noxa, one of the Bak activators. If successful, the proposed experiments will provide new mechanistic insight into the action of a variety of anticancer drugs that activate Bak and into the action of bortezomib.